Human blood is evaluated in vitro for a broad array of diagnostic purposes. Blood is composed of blood cells and plasma. Platelets, which are the smallest of the three major types of blood cells, are only about 20% of the diameter of red blood cells, the most numerous cell of the blood. The normal platelet count is 150,000-350,000 per microliter of blood but since platelets are so small, they make up just a tiny fraction of the blood volume. A principal function of platelets is to maintain homeostasis of blood and prevent bleeding. Platelet function is therefore one indicator of blood homeostasis.
Blood homeostasis refers to the preservation of the bloodstream in an intact and normally functioning manner. This includes maintenance of the chemical properties of blood, and the intactness of blood and vasculature. In the event of an interruption of vascular intactness, for example from a cut, trauma, surgery, or other events that typically cause “bleeding”, a cascade of cellular and biochemical processes within the blood is initiated, with the ultimate goal of preventing or minimizing loss of blood. A visual endpoint of this biological response is the formation of a scab. At a molecular and cellular level, the processes involve interactions between proteins normally circulating in blood and platelets. Several proteins in blood, as well as the platelets themselves, react to exposure to a protein called “tissue factor” which is present in many other tissues throughout the body, but notably is absent from the inside of the veins and arteries comprising normal vasculature. Through direct and indirect chemical pathways, platelets respond to the presence of tissue factor by aggregating, an irreversible (or “one time only”) process by which they dramatically change shape and actively bind each other. This process is known as platelet aggregation. Other enzymes in the blood also react and start to alter proteins in the blood, which start to form insoluble fibrous masses. Analogous to filling a hole with spackle, these insoluble mixtures of proteins, platelets, and other blood components occlude the “hole” in the vascular wall, and, in simple terms, “stop the bleeding”.
More scientifically, when exposed to a damaged blood vessel, platelets will adhere to exposed sub-endothelial matrix. Following the initial adhesion, various factors are released or produced at the site of injury (including thrombin, ADP, growth factors, and collagen) which activate the platelets. Once platelets are activated, a conformational change occurs in the platelet glycoprotein GPIIb/IIIa receptor, allowing it to bind fibrinogen and/or von Willebrand factor. It is believed that this binding of the multivalent fibrinogen and/or von Willebrand factor molecules by GPIIb/IIIa receptors on adjacent platelets results in the recruitment of additional platelets to the site of injury and their aggregation to form a hemostatic plug or thrombus. Platelet aggregation is a term used to describe the binding of platelets to one another. Platelet aggregation is also associated with degranulation, a process through which granules (“envelopes” that contain proteins and small molecules) are released into the surrounding plasma. This process is known as degranulation. These granule contents serve to further accelerate restoration of hemostasis and stimulate cell repair (healing) processes on the vascular wall and any non-vascular tissue.
Without a sufficient number of platelets, or in cases where normal platelet function is impaired or even absent, there is a significant risk of extensive bleeding. Platelet transfusions are administered to patients who have undergone severe trauma, or in cases of emergency surgery where there has been extensive loss of blood.
Understandably, measurements of the ability of platelets to aggregate and thus facilitate or accelerate blood clotting can be important in a number of clinical settings.
It is known that platelet aggregation plays a key role in the pathogenesis of thrombosis and acute coronary artery disease. Evidence suggests that significant variation in inhibition of platelet function exists in the response to various antiplatelet agents. It has also been demonstrated that an inter-individual variability in platelet aggregation exists when P2Y12 antagonists, such as clopidogrel (Plavix), are used for treatment of patients to achieve an anti-aggregation effect. For example, the results of one study demonstrated that at least 10% of patients receiving the drug did not achieve the expected platelet aggregation inhibition (Muller et al., Thromb Haemost. 89(5):783-7 (2003)). Thus, given the acute nature of adverse cardiovascular events, it can be critical to know that the first therapeutic approach selected for a patient will have immediate benefit, ideally without having to monitor the patients and be forced to select alternative therapies. Thus, before patients undergo such therapy, they often have blood samples drawn and tested for platelet function. Similar testing is often employed for pre-surgical screening to rule out potential adverse bleeding effects during surgery/recovery.
It is also desirable to stabilize platelets in drawn blood samples for purposes of testing for disease biomarker testing. Platelets contain proteins and metabolites of diagnostic interest. However, the concentration of the freely circulating forms of these biomarkers in plasma is much more relevant for purposes of diagnosing disease conditions. It is believed that degranulation of platelets, especially upon platelet activation or aggregation, can lead to artificially elevated levels of these markers and represents a preanalytical error if not controlled. The platelet granules also contain enzymes which can catalyze degradation of these circulating biomarkers, and thus result in artificially low levels of the biomarkers of interest.
Further, it is desirable to provide stable platelets for use in therapeutic applications. Autologous platelet gel therapy (which involves isolation of so-called “platelet-rich plasma”) is used to treat certain wounds and a wide range of other conditions ranging from dental implant healing to injections intended to repair ligament damage. If platelets aggregate or become prematurely activated, they may lose this therapeutic effect. As such, there is a need to provide stabilized platelets which could further enhance these processes.
Results of in vitro platelet function can be inaccurate if the platelets are not stabilized in the drawn blood sample, either allowing them to aggregate prior to testing (e.g., no function “left” to test for), or perhaps to “die” or otherwise lose natural function prior to testing. Platelets are inherently unstable in drawn blood, principally because their natural role is to aggregate in response to disruption of the vasculature. Chemical stimulation of platelet aggregation will happen spontaneously at a low level in a drawn blood sample. Over time after the blood is drawn, the platelets aggregate because of this spontaneous stimulation, and so there are fewer and fewer platelets in their original state that are still able to be stimulated whenever the blood sample is finally going to be tested. The current gold-standard of clinical practice calls for platelet function testing to be done on a citrate-anticoagulated blood sample within a maximum of 2 to 4 hours after blood draw (Clinical Laboratory Standards Institute Guideline, “Platelet Function Testing by Aggregometry, H58-A (Vol. 28, No. 31 (2008)). After this time, the sample will have lost much of its original platelet function that it might not be usable for clinical measurements. This widely applied operational standard limits the broad-market utility of platelet function testing. In current practice, for example, many blood samples are sent out from physician's offices to regional testing facilities and may not be tested for many hours or possibly even days after being drawn.
Thus, a need remains for stabilizing blood and blood components such as platelets in compositions such as collected blood samples that better preserves function after collection and during storage or transport, prior to analysis.